Exploiting a subtype-specific mitochondrial vulnerability for successful treatment of colorectal peritoneal metastases

Summary Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.

C Box plots depicting differentially detected metabolites in CMS2/3 vs CMS4 cell lines.Data represent the mean ± SD from CMS2/3 (n=14) vs CMS4 (n=6) cell lines, statistical analysis using unpaired t-test.Data from the CCLE metabolomics dataset 26 .D Box plots depicting differentially expressed proteins between CMS2/3 vs CMS4 patients.Data from the CPTAC proteomics dataset. 27Protein expression (Log2ratio) was calculated as the ratio of sample abundance to reference abundance using the summed reporter ion intensities from peptides that could be uniquely mapped to a gene.The relative abundances were then log2 transformed and zero-centered for each gene to obtain final, relative abundance values.For further details, we refer to the original paper. 27Data represent the mean ± SD, statistical analysis using unpaired t-test.E Dose-response curves of HT55 cell lines treated with ESRRA inhibitor for 72 h.Data are means ± SD of 3 biological replicates, normalized to solvent control.F Relative mitochondrial abundance after 72 h treatment of control or ESRRAi (5 µM), quantified by mtDNA copy number.Data represents the means ± SD of 3 technical replicates.G Cell viability of HT55 cells after 72 h treatment of control, ES-Cu (100 nM), ESRRAi (5 µM), and the combination of ES-Cu (100 nM) and ESRRAi (5 µM).Data are means ± SD of 3 biological replicates, normalized to control.Statistical analysis using unpaired t-test.H Bar graphs depicting the cell viability 72 h after treatment with indicated concentrations of ES in sorted populations using HUTU-80 cells that were transduced with pLV-mitoDsRed (mito LOW and mito HIGH ).Data are means ± SD of three biological replicates.

SUPPLEMENTAL TABLES
Table S1 | Baseline characteristics of HIPEC treated patients in Amsterdam UMC cohort.Related to Figure 1.For categorical variables, numbers and proportions per subgroup were presented.Continuous variables were described by median and range.Continuous variables were compared using Mann-Whitney test.Categorical variables were tested using Chi-square test.R1 = no macroscopic residual tumor, R2a = macroscopic residual tumor smaller than 2.5 mm, R2b = macroscopic residual tumor larger than 2.5 mm Figure S1 | Mesenchymal PDAC cell lines show increased sensitivity to ES treatment.Related to Figure 1.A Kaplan-Meier overall survival analysis of patients treated with CRS-HIPEC with MMC and resection score R1, stratified by CMS2/3 (blue) and CMS4 (green) (log-rank test, p=0.046).B Drug sensitivity of classified PDAC cell line data from GDSC.Shown on x-axis is the relative sensitivity; positive Z-score correlates with high relative sensitivity in mesenchymal cell lines.Y-axis indicates significance (P-value) by t-test of differential sensitivity.Bottom right hand corner includes drugs significantly more effective against mesenchymal cell lines.C Indicated PDAC cell lines representing the mesenchymal and non-mesenchymal subtype were treated for 7 days with ES, after which cells were counted by bead-calibrated flow cytometry.Curves were fitted with nonlinear regression, dose response curves.P-value (by ANOVA) is shown for pooled mesenchymal cell lines (n=4, shown in pink) versus pooled non-mesenchymal cell lines (n=5, shown in grey).

Figure S2 |
Figure S2 | Antioxidants do not prevent cell death following treatment with ES.Related to Figure 2. A Dose-response curves of HUTU-80 and MDST8 cells treated with indicated doses of CuCl2.Cell viability was assessed after 72 h by CellTiter-Blue.Data are means ± SD of 3 biological replicates, normalized to solvent control.B Cell viability was assessed after cells were cultured with regular complete medium (control) or with 5 µM Cu 2+ supplemented to the medium for 72 h.C Cu levels after treatment with 2.5 µM ES-Cu or vehicle, determined by ICP-MS in CMS2 (n=3) and CMS4 (n=3) cell lines.Each dot represents one different cell line.D Caspase-3/7-activation after treatment with 2.5 µM ES + 5 µM CuCl2 at indicated time points (time in h) or vehicle in HUTU-80 and HT55 as determined by flow cytometry.E Superoxide levels after 1 h treatment with 2.5 µM ES + 5 µM CuCl2 or vehicle in HUTU-80 and HT55 as determined by flow cytometry.F Cell viability from ES-Cu treated COLO-320HSR and MDST8 cells incubated with various antioxidants.The data represent the mean of three biological replicates.G Seahorse flux analysis of HUTU-80 cells to measure the real-time oxygen consumption rate (OCR).Regular assay medium (control), 5 µM ES, CuCl2 or the combination of Elesclomol and copper (ES-Cu) was added at t=12 min by injection in the analyzer.In the control no copper was present at all time.Regular mito stress test was applied: oligomycin to inhibit ATP synthesis, FCCP to uncouple the mitochondria, and rotenone/antimycin A to block the mitochondrial electron transport chain.Compounds were added at indicated times.O, oligomycin; F, FCCP; R+A, rotenone with antimycin A. H Cell viability from ES-Cu treated CRC cell lines incubated in galactose or glucose supplemented medium.A fixed concentration of 5 µM CuCl2 was supplemented to the culture medium.The data represent the mean ± SD from 3 biological replicates.Blue represents CMS2 subtype and green represents CMS4.

Figure S3 |
Figure S3 | Relationship between mitochondria, CMS4 and elesclomol sensitivity.Related to Figure 3.A Heatmap representation of the relative expression of oxidative phosphorylation subunits in CMS4 CRC.B Results from Seahorse analysis measuring the baseline ratio of OCR:ECAR in 10 PDAC cell lines, grouped by subtype.Each dot represents an independent cell line.Statistical analysis using unpaired t-test, * p ≤ 0.05.C Box plots depicting differentially detected metabolites in CMS2/3 vs CMS4 cell lines.Data represent the mean ± SD from CMS2/3 (n=14) vs CMS4 (n=6) cell lines, statistical analysis using unpaired t-test.Data from the CCLE metabolomics dataset 26 .D Box plots depicting differentially expressed proteins between CMS2/3 vs CMS4 patients.Data from the CPTAC proteomics dataset.27Protein expression (Log2ratio) was calculated as the ratio of sample abundance to reference abundance using the summed reporter ion intensities from peptides that could be uniquely mapped to a

Figure S4 |
Figure S4 | In vivo treatment with ES-Cu.Related to Figure 4.A IC50 values for Oxaliplatin of CRC cell lines in the GDSC dataset are presented.Each dot represents the IC50 for an individual CRC cell line (n=47).The large whisker represents the mean and the smaller whisker represents the upper SD.B As for panel A, showing Mitomycin C IC50s (n=37).C Immune deficient mice were grafted with HUTU-80 or MDST8 cells.After 2 weeks, mice received a single intraperitoneal 1 ml injection of vehicle control or 25 μM ES-Cu.After an additional 4 weeks, mice were sacrificed and tumor burden was assessed using the mPCI.Statistical analysis using unpaired t-test.

Figure S5 |
Figure S5 | Increased sensitivity of CMS4 cells to ES-Cu in hyperthermia.Related to Figure 5.A IC50 of CMS4 cell lines HUTU-80 and MDST8 following 60 min ES-Cu treatment at 37°C or 42°C.After washing, cells were left to grow for 72 h after which cell viability was assessed by CellTiter-Blue.B Dose-response curve of ES-Cu treated CC531 cells.Cells were treated for 60 min.After washing, cells were left to grow for 72 h after which cell viability was assessed by CellTiter-Blue.C Cells were exposed to 60 min of ES-Cu (2.5 µM), MMC (2.5 µM) and OX (400 µM).After washing, cells were left to grow for 48 h after which cell viability was assessed by CellTiter-Blue.Data are means ± SD of minimally 3 biological replicates.

Table S2 | Baseline characteristics of Eindhoven cohort.
Related to Figure1.For categorical variables, numbers and proportions per subgroup were presented.Continuous variables were described by median and range.Continuous variables were compared using Mann-Whitney test.Categorical variables were tested using Chi-square test.R1 = no macroscopic residual tumor.

Table S3 | Geneset enrichment analysis results from tested genesets.
Related to Figure 3.